基于流式细胞术的保加利亚乳杆菌活性检测方法的应用研究
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摘要
目的采用流式细胞术对现今售卖模式下的含保加利亚乳杆菌饮料中活性菌进行实时监测。方法利用SYTO~?24与碘化丙锭(propidium iodide,PI)双染色法对发酵乳饮料中的保加利亚乳杆菌的活性检测,并对模拟现有售卖模式下发酵乳制品在其验货或销售等常温保存阶段保加利亚乳杆菌活性的变化进行检测。结果同一样品,流式细胞术检测活性保加利亚乳杆菌结果与平板计数法结果在同一数量级内。在商超验货等常温放置时间在5h内,活性保加利亚乳杆菌的数量级不会有显著性变化(P<0.5)。结论该方法能够对保加利亚乳杆菌活性有效检测,且能够缩短单样品的检测时间,适用于商品的快速检测和放行。
Objective To real-time monitor the active bacteria in Lactobacillus bulgaricus beverages in current sales mode by flow cytometry. Methods Activity of Lactobacillus bulgaricus in fermented milk beverage was detected by using SYTO~?24 with propidium iodide(PI) double staining method. The changes of the activity of Lactobacillus bulgaricus in the normal temperature preservation stage such as the inspection or sale of the fermented dairy products under the existing sales model were detected. Results For the same sample, flow cytometry showed that the activity of Lactobacillus bulgaricus was in the same order of magnitude as the plate count method. There was no significant change in the order of magnitude of active Lactobacillus bulgaricus within 5 h at room temperature such as commercial inspection(P<0.5). Conclusion The method can effectively detect the activity of Lactobacillus bulgaricus, shorten the detection time of a single sample, and is suitable for rapid detection and release of commodities.
Objective To real-time monitor the active bacteria in Lactobacillus bulgaricus beverages in current sales mode by flow cytometry. Methods Activity of Lactobacillus bulgaricus in fermented milk beverage was detected by using SYTO~?24 with propidium iodide(PI) double staining method. The changes of the activity of Lactobacillus bulgaricus in the normal temperature preservation stage such as the inspection or sale of the fermented dairy products under the existing sales model were detected. Results For the same sample, flow cytometry showed that the activity of Lactobacillus bulgaricus was in the same order of magnitude as the plate count method. There was no significant change in the order of magnitude of active Lactobacillus bulgaricus within 5 h at room temperature such as commercial inspection(P<0.5). Conclusion The method can effectively detect the activity of Lactobacillus bulgaricus, shorten the detection time of a single sample, and is suitable for rapid detection and release of commodities.
引文
[1]GB 19302-2010食品安全国家标准发酵乳[S].GB 19302-2010 National food safety standard-Fermented milk[S].
[2]尹胜利,杜鉴,徐晨.乳酸菌的研究现状及其应用[J].食品科技,2012,(9):25-29.Yin SL,Du J,Xu C.Research status and application of lactic acid bacteria[J]Food Sci Technol,2012,(9):25-29.
[3]湛剑龙,胡萍,陈韵,等.功能性乳酸菌的应用研究[J].食品安全质量检测学报,2014,(4):1002-1009.Zhan JL,Hu P,Chen Y,et al.Applied research on functional lactic acid bacteria[J].J Food Saf Qual,2014,(4):1002-1009.
[4]李磊,杨雨晗,王双,等.细胞活性检测方法之比较[J].生物学杂志,2011,28(1):87-90.Li L,Yang YH,Wang S,et al.Comparison of cell viability assays[J].JBiol,2011,28(1):87-90.
[5]司徒镇强,吴军正.细胞培养[M].北京:世界图书出版公司,2007.Situ ZQ,Wu JZ.Cell culture[M].Beijing:World Book Publishing Company,2007
[6]肖安风,周祥山,周利,等.应用流式细胞术检测毕赤酵母的细胞活性[J].微生物学通报,2006,33(6):22-26.Xiao AF,Zhou XS,Zhou L,et al.Detection of cell viability of Pichia pastoris by flow cytometry[J].Microbiol Bull,2006,33(6):22-26.
[7]何方洋,万宇平,贾芳芳,等.流式细胞仪在检测食品中微生物的应用研究[J].山东畜牧医,2016,37(8):82-84.He FY,Wang YP,Jia FF,et al.Application of flow cytometry in detection of microorganism in food[J].Shandong J Anim Sci Vet Med,2016,37(8):82-84.
[8]杜耿记,金珠,吴小慧,等.流式细胞仪快速检测酸奶中乳酸菌含量[J].食品安全质量检测学报,2017,8(7):2810-2814.Du GJ,Jin Z,Wu XH,et al.Determination the content of lactic acid bacteria in yoghurt by flow cytometry[J].J Food Saf Qual,2017,8(7):2810-2814.
[9]姜凯.基于流式细胞术的乳酸菌快速计数方法研究和活性判定[D].上海:上海师范大学,2018.Jiang K.Flow cytometry-based rapid counting method for lactic acid bacteria and activity determination[D].Shanghai:Shanghai Normal University,2018.
[10]Anvarian AH,Smith MP,Overton TW.The effects of orange juice clarification on the physiology of Escherichia coli;growth-based and flow cytometric analysis[J].Int J Food Microbiol,2016,219(C):38-43.
[11]陈必成,夏鹏,杨丽红,等.两种荧光染料-CFSE与碘化丙啶染色方法检测细胞杀伤能力[J].中华微生物学和免疫学杂志,2006,26(2):192.Chen BC,Xia P,Yang LH,et al.Detection of cell killing ability by two fluorescent dyes-CFSE and propidium iodide staining[J].Chin JMicrobiol Immunol,2006,26(2):192.
[12]张峻峰,刘道亮,赵占民,等.流式细胞术在微生物快速检测领域的研究进展[J].食品工程,2010,(3):19-22.Zhang JF,Liu DL,Zhao ZM,et al.Progress in flow cytometry in the field of rapid detection of microorganisms[J].Food Eng,2010,(3):19-22.
[13]辛若竹,丁梅,孟宪志,等.活性酸乳中每种益生菌总数的同时计数法[J].食品科学,2008,29(4):244-248.Xin RZ,Ding M,Meng XZ,et al.Simultaneous counting of the total number of probiotics in active yogurt[J].Food Sci,29(4):244-248.
[14]Nevel SV,Koetzsch S,Proctor CR,et al.Flow cytometric bacterial cell counts challenge conventional heterotrophic plate counts for routine microbiological drinking water monitoring[J].Water Res,2017,113:191-206.
[15]陈晓燕.酸奶中乳酸菌计数方法的探讨[J].食品安全导刊,2016,(13):72-73.Chen XY,Discussion on counting method of lactic acid bacteria in yogurt[J].China Food Saf Magaz,2016,(13):72-73.
[16]GB 4789.35-2016食品安全国家标准食品微生物学检验乳酸菌检验[S].GB 4789.35-2016 National food safety standard-Food microbiological analysis-Lactobacillus test[S].
[2]尹胜利,杜鉴,徐晨.乳酸菌的研究现状及其应用[J].食品科技,2012,(9):25-29.Yin SL,Du J,Xu C.Research status and application of lactic acid bacteria[J]Food Sci Technol,2012,(9):25-29.
[3]湛剑龙,胡萍,陈韵,等.功能性乳酸菌的应用研究[J].食品安全质量检测学报,2014,(4):1002-1009.Zhan JL,Hu P,Chen Y,et al.Applied research on functional lactic acid bacteria[J].J Food Saf Qual,2014,(4):1002-1009.
[4]李磊,杨雨晗,王双,等.细胞活性检测方法之比较[J].生物学杂志,2011,28(1):87-90.Li L,Yang YH,Wang S,et al.Comparison of cell viability assays[J].JBiol,2011,28(1):87-90.
[5]司徒镇强,吴军正.细胞培养[M].北京:世界图书出版公司,2007.Situ ZQ,Wu JZ.Cell culture[M].Beijing:World Book Publishing Company,2007
[6]肖安风,周祥山,周利,等.应用流式细胞术检测毕赤酵母的细胞活性[J].微生物学通报,2006,33(6):22-26.Xiao AF,Zhou XS,Zhou L,et al.Detection of cell viability of Pichia pastoris by flow cytometry[J].Microbiol Bull,2006,33(6):22-26.
[7]何方洋,万宇平,贾芳芳,等.流式细胞仪在检测食品中微生物的应用研究[J].山东畜牧医,2016,37(8):82-84.He FY,Wang YP,Jia FF,et al.Application of flow cytometry in detection of microorganism in food[J].Shandong J Anim Sci Vet Med,2016,37(8):82-84.
[8]杜耿记,金珠,吴小慧,等.流式细胞仪快速检测酸奶中乳酸菌含量[J].食品安全质量检测学报,2017,8(7):2810-2814.Du GJ,Jin Z,Wu XH,et al.Determination the content of lactic acid bacteria in yoghurt by flow cytometry[J].J Food Saf Qual,2017,8(7):2810-2814.
[9]姜凯.基于流式细胞术的乳酸菌快速计数方法研究和活性判定[D].上海:上海师范大学,2018.Jiang K.Flow cytometry-based rapid counting method for lactic acid bacteria and activity determination[D].Shanghai:Shanghai Normal University,2018.
[10]Anvarian AH,Smith MP,Overton TW.The effects of orange juice clarification on the physiology of Escherichia coli;growth-based and flow cytometric analysis[J].Int J Food Microbiol,2016,219(C):38-43.
[11]陈必成,夏鹏,杨丽红,等.两种荧光染料-CFSE与碘化丙啶染色方法检测细胞杀伤能力[J].中华微生物学和免疫学杂志,2006,26(2):192.Chen BC,Xia P,Yang LH,et al.Detection of cell killing ability by two fluorescent dyes-CFSE and propidium iodide staining[J].Chin JMicrobiol Immunol,2006,26(2):192.
[12]张峻峰,刘道亮,赵占民,等.流式细胞术在微生物快速检测领域的研究进展[J].食品工程,2010,(3):19-22.Zhang JF,Liu DL,Zhao ZM,et al.Progress in flow cytometry in the field of rapid detection of microorganisms[J].Food Eng,2010,(3):19-22.
[13]辛若竹,丁梅,孟宪志,等.活性酸乳中每种益生菌总数的同时计数法[J].食品科学,2008,29(4):244-248.Xin RZ,Ding M,Meng XZ,et al.Simultaneous counting of the total number of probiotics in active yogurt[J].Food Sci,29(4):244-248.
[14]Nevel SV,Koetzsch S,Proctor CR,et al.Flow cytometric bacterial cell counts challenge conventional heterotrophic plate counts for routine microbiological drinking water monitoring[J].Water Res,2017,113:191-206.
[15]陈晓燕.酸奶中乳酸菌计数方法的探讨[J].食品安全导刊,2016,(13):72-73.Chen XY,Discussion on counting method of lactic acid bacteria in yogurt[J].China Food Saf Magaz,2016,(13):72-73.
[16]GB 4789.35-2016食品安全国家标准食品微生物学检验乳酸菌检验[S].GB 4789.35-2016 National food safety standard-Food microbiological analysis-Lactobacillus test[S].